1. Field of the Invention
The present invention in the field of biochemistry and medicine is directed to modified catalase proteins designed for increased import into peroxisomes and combinations of these with polypeptides that enhance cellular delivery and uptake of proteins. These compositions are used to treat conditions, such as diseases and disorders associated with aging or with peroxisome deficiency and resultant excesses of hydrogen peroxide and other reactive oxygen species.
2. Description of the Background Art
Peroxisomes are essential subcellular organelles of eukaryotic cells. These multifunctional structures arise through the carefully orchestrated reactions of some two dozen proteins, called peroxins (Terlecky and Fransen, 2000). These are critical processes; defects leave cells either devoid of peroxisomes, or with organelles rendered unable to carry out the myriad of biochemical and metabolic functions ascribed to them. Often, such failings result in disease (Gould and Valle, 2000).
Despite major recent advances in an understanding of how the peroxisome arises and functions, only scant information is available regarding the relationship of the organelle and cellular aging. It is unclear, for example, how the organelle functions as cells age, and what role, if any, the peroxisome plays in the aging process.
The present inventors used, as their model system, human diploid fibroblasts (HDFs), cells with a finite replicative lifespan. These somatic cells divide (or double) in culture until they reach a limit referred to as the “Hayflick number” (Hayflick, 1965). At this point, their cell-cycle arrests, and they are termed “senescent.” This process of cellular senescence occurs in aged whole organisms as well (Dimri et al., 1995). Contributing factors to cellular senescence include telomere shortening, DNA damage and related genomic instability, modified gene expression, and the accumulation of reactive oxygen species (ROS) (reviewed in Johnson et al., 1999). With respect to the latter, mitochondria are widely regarded as the chief cellular generators of ROS, and ironically, a major focus of free radical assault (Lee and Wei, 2001; Beckman and Ames, 1998). However, mitochondria are not the only source of cellular ROS.
Another ROS source are the peroxisomes which house, among their constituent enzymes, a variety of hydrogen peroxide (H2O2)-generating oxidases. These organelles also contain catalase, which decomposes H2O2 to water and oxygen and, presumably, prevents accumulation of this toxic compound. Thus, the peroxisome maintains a delicate balance with respect to the relative concentrations or activities of these enzymes to ensure no net production of ROS. How the organelle maintains this equilibrium is unclear, though it is known that peroxisomal pro- and anti-oxidants are tightly coupled, and, under normal conditions, no net accumulation of ROS occurs. It is also not known what happens to these regulatory mechanisms as cells (and organisms) age.
Proteins are directed to the peroxisome by specific peptide sequences, called peroxisomal targeting signals (PTSs), which are recognized by receptor molecules. All but a select few human peroxisomal proteins contain PTS1, a carboxy-terminal sequence (Subramani, 1998). PTS1 is identified and shuttled to the peroxisome by the soluble peroxin, Pex5p (Dammai and Subramani, 2001). For the majority of peroxisomal enzymes, PTS1 is a tripeptide consisting of Ser-Lys-Leu (=SKL) or a closely related variant (Subramani, 1998). In contrast, catalase's PTS1 is a non-canonical PTS1, consisting of the four amino acids, Lys-Ala-Asn-Leu (=KANL)) SEQ ID NO:1) (Purdue and Lazarow, 1996).
As disclosed herein, according to the present invention, these distinct PTS1s lead to dissimilar recognition by Pex5p, with SKL being a far better substrate than KANL (SEQ ID NO:1), and, in aging cells, to significantly different import efficiencies. As disclosed herein, aging fibroblasts produce increasing amounts of ROS as an apparent consequence of this uncoupling of peroxisomal pro- and antioxidants. Finally, the present characterization of peroxisomes in aging cells reveals changes in the size and number of these organelles, as well as in their ability to cycle Pex5p from their surfaces and permit its return to the cytosol.
F. G. Sheikh et al., Proc. Natl. Acad. Sci. USA 95:2961-66, 1998) described human cells which did not import catalase efficiently which were derived from an individual with severe neuropathology. In an effort to restore peroxisomal catalase in these cells, the investigators altered the targeting signal of the enzyme. However, the protein was reintroduced by transient transfection with the corresponding gene. Although this strategy corrected the cells' inability to import catalase, this document provided no detailed analysis of why the approach worked. Moreover, transfection of cells and accompanying (drug) selection for stable transformants is clearly not compatible with the therapeutic approaches of the present invention. Upon inspection of the oligonucleotides used in this study, it is possible to conclude that the genetic constructs produced in this study would encode a catalase protein having at its C terminus the sequence KANL-SLL (SEQ ID NO:21), not even the −SKL tripeptide terminus that the authors ostensibly sought to append to the C-terminus of catalase. Moreover, this study focused solely on restoring catalase in a cell line of one particular patient—but did not disclose what the present inventors have discovered and disclose here for the first time: similar mistargeting of catalase occurs in aging human cells. Obviously, then, the Sheikh et al. document did not even suggest the notion of treating cells prophylactically to slow down aging processes nor the replacement of the native KANL (SEQ ID NO:1) C-terminus of human catalase with the sequences disclosed herein.
Jin et al., Free Radicals Biol Med 31:1509-19, 2001, disclosed that catalase may be introduced into human cells using “protein transduction domains” (PTDs) which are specialized peptide sequences. However, the Jin et al. transduction methodology was not state-of-the-art. A recombinant fusion protein was created, in which the PTD was fused directly to the N-terminus of catalase. The fusion protein was expressed, purified under denaturing conditions, and then added to cells. Given these conditions, the denatured catalase that entered the cells had to refold to its active form. Although the document provides evidence that the enzyme entered cells and processed some ROS, there was no evidence suggesting that the molecule was delivered to the peroxisome—its “correct” intracellular address. Indeed, to the contrary, the findings suggested cytosolic accumulation of the enzyme.
The Jin et al. study lacks any description of the presence, in the primary structure of catalase (or catalase fusion protein) of a strong peroxisomal targeting signal. It should also be noted that others have developed protein transduction domains that permit delivery of proteins without requiring denaturation and, perhaps more importantly, without requiring in-frame fusions with the molecule of interest. The present invention is also directed to such molecules.
Morris et al., Nature Biotech 19:1173-76, 2001, described a protein transduction approach which effectively introduces a protein of interest in its native state. The “carrier” simply dissociates from its ligand once having entered the cell, and no longer appears as part of the equation. The present inventors have conceived of combining this technology with a reengineered catalase molecule as the basis of certain embodiments of this invention.